Effects of low‐dust forages on dust exposure, airway cytology, and plasma omega‐3 concentrations in Thoroughbred racehorses: A randomized clinical trial

Abstract Background Racehorses commonly develop evidence of mild asthma in response to dust exposure. Diets deficient in omega‐3 polyunsaturated fatty acids (Ω‐3) might exacerbate this response. Hypothesis To compare dust exposure, bronchoalveolar lavage fluid (BALF) cytology, and plasma Ω‐3 and specialized pro‐resolving mediators (SPM) concentrations amongst racehorses fed dry hay, steamed hay, and haylage. Animals Forty‐three Thoroughbred racehorses. Methods Prospective clinical trial. Horses were randomly assigned to be fed dry hay, steamed hay, or haylage for 6 weeks. Measures of exposure to dust in the breathing zone were obtained twice. At baseline, week‐3, and week‐6, BALF cytology was examined. Plasma lipid profiles and plasma SPM concentrations were examined at baseline and week 6. Generalized linear mixed models examined the effect of forage upon dust exposure, BALF cytology, Ω‐3, and SPM concentrations. Results Respirable dust was significantly higher for horses fed hay (least‐square mean ± s.e.m. 0.081 ± 0.007 mg/m3) when compared with steamed hay (0.056 ± 0.005 mg/m3, P = .01) or haylage (0.053 ± 0.005 mg/m3, P < .01). At week 6, BALF neutrophil proportions in horses eating haylage (3.0% ± 0.6%) were significantly lower compared with baseline (5.1 ± 0.7, P = .04) and horses eating hay (6.3% ± 0.8%, P < .01). Plasma eicosapentaenoic acid to arachidonic acid ratios were higher in horses eating haylage for 6 weeks (0.51 ± 0.07) when compared with baseline (0.34 ± 0.05, P < .01) and horses eating steamed (0.24 ± 0.02, P < .01) or dry hay (0.25 ± 0.03, P < .01). Conclusions and Clinical Importance Steamed hay and haylage reduce dust exposure compared with dry hay, but only haylage increased the ratio of anti‐inflammatory to pro‐inflammatory lipids while reducing BAL neutrophil proportions within 6 weeks.

anti-inflammatory to pro-inflammatory lipids while reducing BAL neutrophil proportions within 6 weeks.

K E Y W O R D S
airway, asthma, haylage, inflammation, neutrophils, resolution, respiratory tract 1 | INTRODUCTION Exposure to organic dust is central to the development of mild asthma in horses (MA), 1,2 a commonly encountered, 1,3-5 performance-limiting 1,4 inflammatory respiratory disease. Compared with horses at pasture, stabled horses are exposed to higher concentrations of organic dust, 2,6 which includes varying quantities of immunoreactive bacterial and fungal derived products such as endotoxin and β-glucan depending upon the bedding and feed used. 7 In racehorses, bronchoalveolar lavage fluid (BALF) mast cell proportions vary with respirable β-glucan exposure, whereas BALF neutrophil proportions increase with increasing respirable dust exposure, a response modulated by concurrent endotoxin exposure. 1 Racehorses remain in their stall an average of 23 hours/day and are mainly fed dry hay. Other forages with higher moisture content and lower dust production are available. Haylage, a conserved forage that is harvested at a higher moisture content than hay, reduces exposure of horses to respirable dust by 60% to 70% when compared with dry hay. 8,9 Alternatively, dry hay can be steamed to reduce respirable dust release in vitro by 95%, 10 and bacterial and mold content by 99%, 10,11 Outside of the laboratory, the efficacy of low-dust forages to reduce dust exposure and airway inflammation remains unknown.
Resolution of inflammation is often prolonged and incomplete once low-dust management is instituted. [12][13][14] Chronic inflammation present in the asthmatic airways might result not only from repetitive exposure to triggering stimuli but also from impaired pro-resolving pathways. 13,14 Specialized pro-resolving mediators (SPM) play an active role in the return to tissue homeostasis after an inflammatory event by reducing leukocyte recruitment, inducing neutrophil apoptosis, and enhancing efferocytosis at the site of inflammation. [15][16][17][18] SPM are derived from essential omega-3 (Ω-3), and to a lesser extent, omega-6 (Ω-6) polyunsaturated fatty acids (PUFAs) 19 ; the relative availability of each are determined by dietary intake. Arachidonic acid and its inflammatory derivatives, prostaglandins, thromboxane, and leukotrienes, are derived from Ω-6, and excessive Ω-6 intake has been associated with increased inflammatory diseases in people. 20 Increased intake of Ω-3 might help mitigate airway inflammation in humans 21 and horses with severe asthma. 12 Racehorses are typically fed dry hay, which contains less α-linolenic acid (Ω-3) when compared with pasture and haylage. 22 Therefore, in addition to lowering dust exposure, feeding haylage to racehorses might provide further benefit because of higher Ω-3 content.
Consequently, we hypothesized that feeding haylage or steamed hay to racing Thoroughbreds in place of dry hay would reduce breathing-zone measures of respirable dust, β-glucan, and endotoxin, and would result in significantly lower BALF proportions of neutrophils and mast cells. We expected that horses fed haylage would have higher plasma concentrations of Ω-3 and SPMs and display faster resolution of airway inflammation.

| Experimental design
A prospective randomized feed trial was designed to compare respirable dust exposure and airway cytology between horses fed dry hay, haylage, or steamed hay for 6 weeks. The study was performed during two racing meets between May 2018 and October 2019. Trainers allowed horses to enroll in the study by providing informed consent and completing a short questionnaire detailing the length of ownership, vaccination history, and any history of respiratory illness. Horses with signs of respiratory infection or systemic illness (fever, abnormal hematology, decreased appetite) were excluded. Upon enrollment, horses were allocated to receive dry hay, haylage, or steamed hay as their sole source of forage using simple randomization through computer-generated random numbers. Vaccination against Clostridium botulinum type-B before assignment to haylage was offered to the trainer. No other change was made to the horses' management.
Horses continued to be fed the same amount of grain, mineral, and vitamin supplements according to the trainer's preference. All horses were bedded on sawdust. Horses fed hay and steamed hay were eligible for re-enrollment.
At baseline, physical examination, blood collection, endoscopy of the respiratory tract, and bronchoalveolar lavage (BAL) were performed. For those horses assigned to receive haylage, the forage was gradually introduced whereas hay was gradually excluded from the diet over the course of 7 days. Those horses assigned to dry and steamed hay groups continued to be fed the hay they received before enrollment. Trainers were provided with haylage (C&M Forage) and with a commercially available hay steamer (Haygain) free of charge and instructed on its use. Haylage with visible mold growth was discarded, and trainers were instructed to feed haylage bales within 3 days once the plastic wrap was opened. Horses remained on the assigned forage for 6 weeks. After 3 and 6 weeks on the assigned forage, breathing zone measures of respirable dust exposure, physical and endoscopic examinations, and BAL were performed. During the study, training and racing schedules were continued as usual while adhering to recommended drug elimination times 23 for sedation and topical lidocaine used for the BAL procedure.

Sampling and analysis of respirable dust
Exposures to respirable dust were assessed using methods as described. 24 Briefly, a conventional respirable sampler consisting of a respirable cyclone (P225-01-02, SKC, Inc., Eighty-Four, PA) to remove dust larger than the respirable fraction and was fitted to a The average of three weights of the filter before sampling was subtracted from the average of three weights of the filter after sampling. The weight of respirable dust lighter than 0.02 μg was considered below the limit of detection (LOD). 25 A time-weighted average concentration was calculated by dividing the weight of respirable dust by the sampling volume (=sampling flow rate Â sampling time). The lower LOD for respirable dust concentration was 0.047 mg/m 3 . Subsequently, polyvinyl chloride filters were stored at À20 C until elution for β-glucan and endotoxin analyses.

Real-time measurement of particulate matter
Concentrations of particulate matter (PM) in the horse's breathing zone were measured in real-time using a PM monitor (OPC-N2, Alphasense, Essex, UK). The PM monitor was secured to the crown piece of a break-away halter and its inlet was connected to flexible tubing that extended to the noseband of the halter to sample PM in the breathing zone of the horse. PM concentrations were classified by their size. Specifically, mass concentrations of PM 1 (≤1 μm), PM 2.5 (≤2.5 μm), PM 2.5-10 (from 2.5 to 10 μm), and PM 10 (≤10 μm) were obtained. During the 3-hour measurement period, the horse was free to move around the stall, eat, and drink as usual.

| Βeta-glucan and endotoxin analysis
The content of β-glucan and endotoxin in respirable dust was mea-

| Clinical score
Upon physical examination, a clinical score (range: 0-21) based on cough, nasal discharge, respiratory efforts, and auscultation was determined as described. 12

| Endoscopic examination
Horses were restrained with a nose twitch and a flexible videoendoscope with 7.9 mm outer diameter was passed through the ventral meatus to the level of the pharynx. A score was assigned to the degree of pharyngeal lymphoid hyperplasia present from 0 (no follicles) to 4 (numerous, large, edematous follicles). 2 Any upper respiratory tract abnormality was noted. Tracheal mucus was scored from 0 (no mucus) to 4 (large, pool-forming). 26 To facilitate BAL, the carina and larynx were sprayed with a 0.4% lidocaine solution as the endoscope was removed (20-30 mL at each site).

| Bronchoalveolar lavage
Horses were sedated by intravenous injection of xylazine hydrochloride (0.2-0.5 mg/kg; AnaSed, Akorn Animal Health, Lake Forest, IL) and butorphanol (0.02-0.04 mg/kg; Torbugesic, Zoetis, Parsippany-Troy Hills, NJ). A sterile BAL tube (300 cm long; 10 mm OD; Bivona Medical Technologies, Gary, IN) with an inflatable cuff was passed through the nose and wedged into a peripheral bronchus. Five 50 mL aliquots of sterile 0.9% sodium chloride solution were infused and recovered manually using 60 mL syringes. The BALF was filtered with gauze and immediately placed on ice. Samples were processed within 1 hour of collection. Cytological specimens were prepared by cytospin centrifugation and processed with modified Wright's stain. Differential cell counts were performed on 600 cells by a single observer (CO), including at least five microscopic fields 27 ; epithelial cells were not included in the cells counted. Threshold values were used to determine the presence of airway inflammation in BALF were neutrophils >5%, mast cells >2%, or eosinophils >1%. 3

| Fatty acid analysis
Venous blood was collected from the jugular vein into evacuated tubes containing EDTA for hematology at baseline and plasma separation at baseline, week-3 and week-6. Plasma samples were stored at À80 C until analysis. Fatty acid analysis of plasma samples was performed on freshly thawed aliquots after extraction of lipids by the Folch method, 28 isolation of phospholipids by solid phase extraction using silica cartridges, and methylation utilizing boron trifluoride and gas chromatography. 29 Fatty acid measured included linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

| Specialized pro-resolving mediator analysis
Plasma samples from baseline and week-6 were stored at À80 C until analyses. Selected SPM were measured using equine-specific ELISA kits for resolving D1 (RvD1) and resolving E1 (RvE1; MyBiosource, San Diego, CA) according to the manufacturer's instructions, using a plate reader (BioTek, Winooski, VT). Measures were performed in duplicate, and the average was recorded.

| Sample size calculations
Preliminary data demonstrated a mean BALF neutrophil proportion of 4.75% (SD = 3.95%) in horses racing at the racetrack 1 and a mean BALF neutrophil proportion of 0.75% (4-fold reduction from baseline) in horses fed haylage for 6 weeks. 9 A sample size of 15 horses per group, or 45 total horses, would provide a 90% power to detect a statistically significant difference between groups with P ≤ .05. To allow for subject dropout over the 6-week enrollment period, a target enrollment of 20 per group for a total of 60 horses was planned.

| RESULTS
Data were reported according to the recommendations from the 2010 CONSORT statement (http://www.consort-statement.org/). Raw data are reported as mean ± SD, while results of statistical models are reported as least-square means ± SE.

| Horses
Forty-three horses were enrolled in the study. In response to inadequate enrollment, horses randomly assigned to be fed dry or steamed hay were eligible for re-enrollment. Horses randomly assigned to be fed haylage were not eligible for re-enrollment within the calendar year because of unknown duration of potentially altered PUFA profiles. This enrollment strategy led to re-enrollment of 21 horses, with 12 horses re-enrolled once, and 9 horses re-enrolled twice. Seventythree measurements were performed at baseline, 69 at week-3, and 53 at week-6 ( Figure 1, Table S2). Thirteen horses left the barn for causes not related to the study, four could not be sampled because of conflicts with the barn's schedule, and three horses did not receive the assigned forage (steamed hay) because of an error operating the hay steamer. Five stallions (12%), 20 geldings (47%), and 18 mares (42%) were enrolled, and the age was 4.0 ± 1.7 years (mean ± SD).

| Effect of forage on clinical score and tracheal mucus
There was a significant change in clinical score over time regardless of forage assignment (P = .03; Figure 5A). Forage assignment had no effect on the clinical score over time (P = .98). Respiratory rate was not affected by time (P = .60) or forage over time (P = .66).
Mucus score was significantly higher in horses eating hay at week-6 when compared with baseline (P = .04) and week-3 (P = .04; Figure 5B). In addition, mucus score was significantly higher at week-6 in the horses eating hay than those eating haylage (P = .05; Figure 5B).

| Effect of forage on plasma PUFAs and SPMs
No effect of forage or time was observed on plasma levels of linoleic acid, γ-linolenic acid, α-linoleic acid, steridonic acid, eicosadienoic acid, dihome-γ-linolenic acid, arachidonic acid, docosadienoic acid, DHA, or EPA (P > .1; Table 2). The ratio of EPA to arachidonic acid was significantly higher in horses eating haylage at week-6 when compared with baseline (P = .007), and with horses fed hay and steamed hay at week-6 (P = .002 and P = .001, respectively). No effect of time,   since eosinophilic inflammation appears to be more frequent in younger horses. 2 None of the horses that withdrew from the study evidenced any adverse effect from either the feeding or sampling protocol. Haylage and steamed hay were well accepted, and no adverse effects or changes in fecal consistency were observed during the study. Botulism is associated with feeding poorly preserved haylage to horses. 30 We advised horsemen to discard haylage with visible mold growth, and haylage bales were fed within 3 days of opening. 31,32 In addition, vaccination against Clostridium botulinum type B was offered free of charge. The hygienic quality of both hay and haylage can vary widely depending on composition and conditions during production, 33 Average PM 10 and PM 10-2.5 exposures were lower for the horses eating haylage when compared with steamed hay and hay. Also, PM 10 was significantly lower in horses eating steamed hay when compared with horses eating hay. In racehorses, higher exposure to breathing zone PM 10 is associated with increased tracheal mucus accumulation and higher neutrophil proportions in tracheal wash. 38 In humans, increased PM 2.5 and PM 10 exposures are associated with exacerbation of asthma. 39 In addition, PM 10-2.5 exposure is related to the development of asthma in children, 40   Clinical score changed over time independent of forage, but the changes were small, <0.5 points on a 21-point scale, and not considered clinically relevant. Furthermore, the clinical score at baseline was not different from the score at week-6.

| DISCUSSION
Endoscopic mucus score increased over time when horses were eating hay, and the mucus score of horses fed haylage at week-6 was lower than horses fed hay. While mucus scores are affected by the time elapsed between exercise and endoscopy, 43  In horses with severe asthma, the addition of an Ω-3 supplements to a low dust diet achieves faster improvement in clinical signs and BALF cytology compared with horses fed a low-dust diet with placebo. 12 Furthermore, plasma DHA concentrations are significantly higher in the horses fed algae-derived Ω-3 supplement. In humans, diets with higher content of DHA and EPA might be protective against inflammatory diseases like asthma while other studies do not find this association. 49 EPA competes with arachidonic acid as a substrate for enzymes and is converted to SPM, 50 and in vitro studies of alveolar macrophages from humans with asthma reveal that EPA is a more potent inhibitor of pro-inflammatory mediators than DHA. 51 Furthermore, EPA is an important precursor of the resolvin E-series of SPM, 46  0.51 ± 0.07* , ** , *** *Different from baseline (P = .007). **Different from horses fed hay at week-6 (P = .002). ***Different from horses fed steamed hay at week-6 (P = .001).
bronchoconstriction associated with a decrease in circulating inflammatory cytokines in vitro and production of leukotrienes from neutrophils in vitro. 52 A limitation of our study was that most of the horses in the study were provided by a single trainer. Compliance with the study design required cooperation and extra work hours from staff that some trainers found burdensome, which limited their participation. In addition, it was impossible to mask investigators to forage assignment.
While this information could potentially bias investigators, the measures of exposure, PUFA, and SPM are objective. BALF differential cell counts might be more vulnerable, as bias can be introduced in selection of cells to enumerate. However, counting at least 600 cells in a systematic manner for each BALF was performed to minimize the risk of such bias.